Membrane ion channels are critical for proper electrolyte transport and fluid balance. The epithelial sodium channel (ENaC) resident to the luminal plasma membrane of electrically tight epithelia, such as that lining the distal renal nephron, is, in particular, essential to regulation of sodium balance. ENaC plays an active role in the maintenance of fluid and blood pressure levels as seen with loss of function and gain of function genetic disorders, Liddle's syndrome and Pseudohypoaldosteronism type I, respectively. ENaC is a pore forming membrane protein composed of three homologous but distinct subunits. The current proposal is designed to improve our understanding of ENaC's structure features by examining the following aims: Aim 1: Residues and motifs that are critical for maintaining ENaC structure and function will be identified through an innovative yeast screen of ENaC function recently developed. Random mutation of targeted regions within ENaC subunits will be generated. After subsequent expression of mutated ENaC subunits in yeast we will identify a set of ENaC loss/gain in function mutations appropriate for continued studies. This sub-set of potential critical residues will be used to generate plausible hypotheses of structure-function relationships, which will be examined in a mammalian expression system using classical electrophysiology in a more mechanistic approach. Aim 2: Potentially critical interactions between ENaC subunits will be identified using surface plasmon resonance analysis of binding interactions. Binding of peptide fragments predicted to be essential for maintaining intersubunit interactions will be investigated first, followed by potential interacting regions identified in Specific Aim 1. The influence of immobilization environment on the observed binding interactions will be determined. We expect that quantification of binding interactions will lead to a greater understanding of critical intersubunit contacts.